Difference between revisions of "Fugene Transfection of 293T/COS Cells"

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#Warm OptiMEM, COS-FBS Media and PBS -/-.
 
#Warm OptiMEM, COS-FBS Media and PBS -/-.
 
#Calculate amount of Fugene needed.
 
#Calculate amount of Fugene needed.
*Per ug of DNA need 3 uL Fugene.
+
##Per ug of DNA need 3 uL Fugene.
*Per uL of Fugene need 16 uL of OptiMEM.
+
##Per uL of Fugene need 16 uL of OptiMEM.
 
#Add Fugene to OptiMEM, incubate ~5 min.
 
#Add Fugene to OptiMEM, incubate ~5 min.
 
#Add required amount of DNA to eppendorf tubes.
 
#Add required amount of DNA to eppendorf tubes.
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#Incubat 5-20 min.
 
#Incubat 5-20 min.
 
#Split confluent cells 2-3X into fresh dishes as follows:
 
#Split confluent cells 2-3X into fresh dishes as follows:
#Wash confluent COS cell plate(s) twice with 10 mL D-PBS -/-
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##Wash confluent COS cell plate(s) twice with 10 mL D-PBS -/-
#Add 1 mL Trypsin solution (0.05%) and incubate at 37C until cells are detached
+
##Add 1 mL Trypsin solution (0.05%) and incubate at 37C until cells are detached
#Add 25 mL COS/FBS
+
##Add 25 mL COS/FBS
#Aliquot cells into wells as required (1 mL per 12well, 2 mL per 6 well)
+
##Aliquot cells into wells as required (1 mL per 12well, 2 mL per 6 well)
 
#Add DNA/Fugene/DMEM to cells
 
#Add DNA/Fugene/DMEM to cells
 
#Leave mixture on Cells for 24-48h to allow protein to accumulate
 
#Leave mixture on Cells for 24-48h to allow protein to accumulate

Revision as of 14:35, 8 June 2009

Protocol

  1. Warm OptiMEM, COS-FBS Media and PBS -/-.
  2. Calculate amount of Fugene needed.
    1. Per ug of DNA need 3 uL Fugene.
    2. Per uL of Fugene need 16 uL of OptiMEM.
  3. Add Fugene to OptiMEM, incubate ~5 min.
  4. Add required amount of DNA to eppendorf tubes.
  5. Add 51 uL/ug OptiMEM/Fugene to each DNA tube.
  6. Incubat 5-20 min.
  7. Split confluent cells 2-3X into fresh dishes as follows:
    1. Wash confluent COS cell plate(s) twice with 10 mL D-PBS -/-
    2. Add 1 mL Trypsin solution (0.05%) and incubate at 37C until cells are detached
    3. Add 25 mL COS/FBS
    4. Aliquot cells into wells as required (1 mL per 12well, 2 mL per 6 well)
  8. Add DNA/Fugene/DMEM to cells
  9. Leave mixture on Cells for 24-48h to allow protein to accumulate